Method for gene diagnosis of bovine Hsp70 deficiency

ABSTRACT

A method for gene diagnosis of bovine Hsp70 deficiency, which comprises (a) a step of obtaining a bovine nucleic acid sample, (b) a step of subjecting the nucleic acid sample obtained in step (a) to a gene amplification reaction to obtain a nucleic acid fragment in which a region including a mutation site likely to be present in bovine Hsp70 gene is amplified, and (c) a step of examining the presence of mutation on the nucleic acid fragment in step (b), the region including the mutation site being a region including 1997-11030 position of a base sequence shown in SEQ ID No. 1 of SEQUENCE LISTING in a base sequence of bovine Hsp70 gene. According to the invention, bovine Hsp70 deficiency and its carrier can be detected and diagnosed by a genetic engineering method easily and quickly, and a kit used for this purpose is provided.

FIELD OF THE INVENTION

[0001] The present invention relates to a method for gene diagnosis (or detection) of bovine Hsp70 deficiency.

[0002] In the invention, “Hsp70” means a protein formed by transcribing and translating Hsp70 gene, and “Hsp70 gene” means a DNA region including an exon of a translated region encoding Hsp70, an exon of an adjacent untranslated region, introns of these regions, a region participating in control of expression of the gene and a mutant portion related with the disease.

BACKGROUND OF THE INVENTION

[0003] The Hsp70 deficiency is an autosomal recessive hereditary disease which is mainly a diaphragmatic muscle disease, clinically showing tympania, respiratory insufficiency and the like. In this disease, muscle fiber denaturation and a core-like structure are observed in a diaphragmatic muscle as histopathological characteristics. The attack of this disease has been found in a Holstein since 1994, while no such disease is reported in humans.

[0004] Since bovine attacked with Hsp70 deficiency repeats tympania, the disease can be found. However, with respect to bovine being a heterozygote carrying a gene related with abnormality only on one chromosome, namely bovine being genetically a carrier of Hsp70 deficiency, it is difficult to find the abnormality.

[0005] Accordingly, even in case of mating a bovine pair with abnormality unobserved apparently, abnormality sometimes appears in a calf to be born. Thus, such a case is problematic in view of preventing attack of the disease.

[0006] In order to prevent bovine Hsp70 deficiency, a method in which mating of heterozygotes is avoided is considered. However, for enabling this method, it is required that diagnosis for bovine Hsp70 deficiency is performed at a gene level to specify a carrier of a disease gene. If it is possible to provide a method which can clarify how a bovine disease gene having abnormality is mutated in comparison to a normal bovine gene and rapidly detect a mutant gene by various genetic engineerings, the genetic diagnosis can be established.

SUMMARY OF THE INVENTION

[0007] Accordingly, it is an object of the invention to provide a method for gene diagnosis (detection) of bovine Hsp70 deficiency. This makes it possible to prevent attack of the disease in future by screening a carrier of Hsp70 deficiency.

[0008] The present inventors have conducted investigations for achieving the foregoing object, and have consequently succeeded in finding deficiency of DNA ranging approximately 11 kb closely related with the disease. The deficient region includes Hsp70 gene already reported as to bovine (M. D. Groz et al., Genomics, 14, 863-868 (1992); J. A. Gutierrez et al., Biochem. J., 305, 197-203 (1995)).

[0009] Further, the present inventors have found that the cause of this disease is that Hsp70 is not expressed owing to mutation, i.e. DNA deficiency, and that the mutation is detected by PCR (polymerase chain reaction) using oligonucleotide primers including specific oligonucleotide primers set in the gene. These findings have led to the completion of the invention.

[0010] The invention and the embodiments thereof are described below.

[0011] The invention is described below.

[0012] (1) A method for gene diagnosis of bovine Hsp70 deficiency, which comprises the following steps,

[0013] (a) a step of obtaining a bovine nucleic acid sample,

[0014] (b) a step of subjecting the nucleic acid sample obtained in step (a) to a gene amplification reaction to obtain a nucleic acid fragment in which a region including a mutation site likely to be present in bovine Hsp70 gene is amplified, and

[0015] (c) a step of examining the presence of mutation in the nucleic acid fragment in step (b),

[0016] the region including the mutation site being a region including 1997-11030 position of a base sequence shown in SEQ ID No. 1 of SEQUENCE LISTING in a base sequence of bovine Hsp70 gene.

[0017] (2) The method for gene diagnosis described in (1), wherein the gene amplification reaction is conducted by a polymerase chain reaction method.

[0018] (3) The method for gene diagnosis described in (1) or (2), wherein the presence of mutation is examined by examining a gene amplification product obtained by the polymerase chain reaction method.

[0019] (4) The method for gene diagnosis described in any one of (1) to (3), wherein the nucleic acid sample is a sample containing genomic DNA, cDNA or mRNA.

[0020] (5) A method for gene diagnosis of bovine Hsp70 deficiency, which comprises conducting genome linkage analysis of subject bovine, isolating bovine Hsp70 gene by positional cloning, determining a base sequence of the gene by a usual manner, and examining the presence or absence of mutation by comparing said base sequence with a base sequence of cDNA encoding normal bovine Hsp70 as shown in SEQ ID No. 1 of SEQUENCE LISTING.

[0021] (6) A kit for detecting bovine Hsp70 deficiency, which kit contains oligonucleotide primers used for amplifying a region including a mutation site likely to be present in bovine Hsp70 gene by a gene amplification reaction, the oligonucleotide primers being selected from the group consisting of

[0022] <1> oligonucleotides having a base sequence corresponding to a 5′-terminal region in a base sequence shown in SEQ ID No. 1 of SEQUENCE LISTING, and

[0023] <2> oligonucleotides having a complementary base sequence to a 3′-terminal region in the base sequence shown in SEQ ID No. 1 of SEQUENCE LISTING.

[0024] (7) The kit described in (6), wherein the oligonucleotide primers comprise from 15 to 35 nucleotides.

[0025] (8) The kit described in (6), wherein the oligonucleotide primers are a pair of oligonucleotide primers selected from the group consisting of those shown in SEQ ID Nos. 2 to 8 of SEQUENCE LISTING, provided combinations of SEQ ID Nos. 2 and 4, 3 and 5, and 6 and 7 are excluded.

[0026] (9) A base sequence which is a whole or a part of a base sequence corresponding to bovine Hsp70 gene or bovine Hsp70 deficiency gene or its complementary chain, and which is selected from the group consisting of

[0027] (a) a whole or a part of a base sequence shown in SEQ ID No. 1 of SEQUENCE LISTING or its complementary chain,

[0028] (b) all sequences which can be used for hybridization to the sequence (a) and gene amplification of a region including a mutation site likely to be present in bovine Hsp70 gene by PCR, and

[0029] (c) sequences derived from the sequences (a) and (b) for degeneracy of a gene code.

[0030] (10) The base sequence described in (9), which is selected from the group consisting of a genomic DNA sequence, a cDNA sequence, an RNA sequence, a hybrid sequence, a synthetic sequence and a semi-synthetic sequence.

[0031] (11) A nucleotide probe which can be hybridized to the base sequence described in any of (9) and (10) or the corresponding mRNA.

[0032] (12) A method for detection of bovine Hsp70 deficiency, which comprises a step of clarifying and/or isolating a sequence including a mutation site likely to be present in bovine Hsp70 gene using the nucleotide probe described in (11).

[0033] (13) The oligonucleotide primers described in any of (6) to (8).

[0034] (14) A gene diagnostic reagent for bovine Hsp70 deficiency, containing the oligonucleotide primers described in (13).

BRIEF DESCRIPTION OF THE DRAWINGS

[0035]FIG. 1(a) is a view showing a deficient portion of bovine Hsp70 gene derived from Hsp70 deficiency-attacked bovine, and FIG. 1(b) is a view showing positions of PCR primers for detecting the deficient portion.

[0036]FIG. 2(a) is an electrophoresis pattern view showing amplification by PCR using genomic DNA free from mutation of Hsp70 as a template for detection of Hsp70 normal type, and FIG. 2(b) is an electrophoresis pattern view showing amplification by PCR using genomic DNA with mutation of Hsp70 as a template for detection of Hsp70 mutant type.

DETAILED DESCRIPTION OF THE INVENTION

[0037] The invention is described in detail below.

[0038] The invention described in (1) is a method for gene diagnosis (detection) of bovine Hsp70 deficiency.

[0039] As will be later described, when the mutation of gene which causes bovine Hsp70 deficiency is clarified (FIG. 1(a)) the detection of the disease can be performed using this mutation. The specific method for gene diagnosis (detection) of bovine Hsp70 deficiency comprises the following steps,

[0040] (a) a step of obtaining a bovine nucleic acid sample,

[0041] (b) a step of subjecting the nucleic acid sample obtained in step (a) to a gene amplification reaction to obtain a nucleic acid fragment in which a gene including a mutation site likely to be present in bovine Hsp70 gene is amplified, and

[0042] (c) a step of examining the presence of mutation in the nucleic acid fragment in step (b).

[0043] First, step (a) is described. The bovine nucleic acid sample used in the invention is not particularly limited so long as it has a base sequence encoding Hsp70. It includes nucleic acids derived from appropriate cells or tissues (including cDNAs transcribed from all genomic DNAs and all RNAs of cells), such as genomic DNAs, cDNAs and mRNAs. The bovine nucleic acid sample can be prepared by a known method, for example, the method described in Molecular Cloning, A Laboratory Manual (2nd edition) (J. Sambrook, E. F. Fritsch & T. Maniatis (Ed.), Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989)).

[0044] Second, step (b) is described. The region including the mutation site likely to be present in bovine Hsp70 gene is amplified by using the nucleic acid sample obtained in step (a) and appropriate primers, whereby a desired nucleic acid fragment can be obtained.

[0045] The method for the gene amplification reaction used in this step is not particularly limited so long as it can amplify the very region. For example, an PCR method, a nucleic acid amplification method using RNA polymerase and a chain substitution amplification method can be used. Of these, the PCR method is preferable.

[0046] The mutation site-including region to be amplified is not particularly limited so long as it includes mutation that causes bovine Hsp70 deficiency in the base sequence of bovine Hsp70 gene. For example, a region including 1997-11030 position in the base sequence shown in SEQ ID No. 1 of SEQUENCE LISTING is mentioned.

[0047] In the present specification, “polymerase chain reaction” or “PCR” generally refers to the method described in U.S. Pat. No. 4,683,195, for example, a method for enzymatically amplifying in vitro a desired base sequence.

[0048] In general, the PCR method includes repeating a cycle of primer elongation (primer extension and synthesis) by using two oligonucleotide primers capable of being hybridized to a template nucleic acid preferentially. As typical primers used in the PCR method, primers complementary to a base sequence to be amplified in a template are available. For example, primers complementary to the base sequence to be amplified at both ends or primers adjacent to the base sequence to be amplified are preferably used.

[0049] PCR can be conducted by the method described in M. A. Innis, D. M. Gelfaud, J. J. Snindky, & T. J. White (Ed.), PCR protocols: A Guide To Methods And Applications, Academic Press, Inc., New York (1990); M. J. McPherson, P. Quirke & G. R. Taylor (Ed.), PCR: A Practical Approach, IRL Press, Oxford (1991); R. K. Saiki et al., Science, 239, 487-491 (1988); M. A. Frohman et al., Proc. Natl. Acad. Sci. USA, 85, 8998-9002 (1988), or its modified or altered method.

[0050] Further, the PCR method can be performed by using an appropriate commercial kit and also according to protocols given by kit makers or distributors.

[0051] In the present specification, “oligonucleotide” is a relatively short single-stranded or double-stranded chain polynucleotide. Polydeoxynucleotide is preferable, and it can chemically be synthesized by a known method such as a triester method, a phosphite method, a phosphoamidite method or a phosphonate method as described in Agnew. Chem. Int. Ed. Engl., Vol. 28, pp. 716-734 (1989). It is commonly known that the synthesis can conveniently be conducted on a modified solid support. For example, it can be conducted with an automated synthesizer which is commercially available. The oligonucleotide may contain one or more modified bases. For example, it may contain a base unusual in nature, such as inosine, or a tritylated base.

[0052] The primers used in the PCR method are not particularly limited so long as they can amplify the DNA fragment including the mutation site. As typical primers, (a) oligonucleotides having a base sequence corresponding to an optional region in a base sequence shown in SEQ ID No. 1 of SEQUENCE LISTING and

[0053] (b) oligonucleotides having a complementary base sequence to an optional region in the base sequence shown in SEQ ID No. 1 of SEQUENCE LISTING can be used. Preferably, (1) oligonucleotides having a base sequence corresponding to a 5′-terminal optional region in the base sequence shown in SEQ ID No. 1 of SEQUENCE LISTING and (2) oligonucleotides having a complementary base sequence to a 3′-terminal optional region in the base sequence shown in SEQ ID No. 1 of SEQUENCE LISTING can be used. Examples thereof include oligonucleotides containing from 3 to 100, preferably from 10 to 50, more preferably 15 to 35 nucleotides.

[0054] The PCR conditions are not particularly limited either, and known PCR conditions are available. The conditions can be selected by referring to the descriptions of the foregoing documents. In PCR, a cycle of thermal denaturation of DNA chain, annealing of primers and synthesis of complementary chain with polymerase is repeated, for example, from 10 to 50 times, preferably from 20 to 35 times, more preferably from 25 to 30 times.

[0055] Third, step (c) is described. In this step, the presence of mutation is examined on the nucleic acid fragment obtained in step (b). The method for detecting the presence of mutation is not particularly limited, and the presence of mutation is detected by examining a length of the DNA fragment obtained by the PCR method.

[0056] A method for examining the length of the DNA fragment is not particularly limited. Preferable is a method in which a DNA fragment is separated by electrophoresis on a polyacrylamide or agarose gel and the desired DNA fragment is identified on the basis of, for example, its mobility relative to a mobility of a marker DNA fragment having a known molecular weight.

[0057] As a detecting method other than the above method, there is a method in which the foregoing step (c) is replaced with another mutation detecting method. For detecting the mutation, a known method for detecting mutation, such as a hybridization method using an appropriate DNA fragment including a mutation site as a probe, can be used. Moreover, the mutation can be detected by a method in which a base sequence is determined by cloning an amplified DNA in an appropriate vector or a method in which by using an amplified fragment per se as a template, its base sequence is determined.

[0058] It is advisable that oligonucleotides or a probe is labeled with a label component for facilitating the detection. The label component can be detected by a spectroscopic method, an optical method, a biochemical method, an immunological method, an enzyme chemical method or a radiochemical method. Examples of the label component include enzymes such as peroxidase and alkaline phosphatase, radioactive labels such as ³²P, isotopes, biotin, fluorochrome, luminous substances and coloring matters.

[0059] It is possible to detect and diagnose not only bovine attacked with Hsp70 deficiency but also bovine being a carrier of Hsp70 deficiency by the method for gene diagnosis (detection) of bovine Hsp70 deficiency of the invention.

[0060] Accordingly, the bovine Hsp70 deficiency of the invention means the genetic abnormality, and it is interpreted in a broad sense by including a carrier, regardless of the presence or absence of the disease.

[0061] Analysis of Hsp70 gene of normal bovine and Hsp70 deficiency-attacked bovine:

[0062] For examining a relation between mutation in a gene and the disease, first, a gene (cDNA) encoding normal bovine Hsp70 is isolated, and its base sequence is clarified. Any example of isolating the very gene has not been reported as yet. The present inventors performed genome linkage analysis of a family including the disease-attacked bovine, and isolated bovine Hsp70 gene by a positional cloning method. That is, it was performed by mapping a causal gene locus on a linkage map and then isolating the causal gene from the chromosomal region (“Dobutsu Iden Ikushugaku Jiten”, published by Japan Livestock Technology Association).

[0063] The whole base sequence of cDNA encoding normal bovine Hsp70 clarified in the invention is shown in SEQ ID No. 1 of SEQUENCE LISTING. Determination of the base sequence of the DNA fragment (sequencing) can be performed by a chemical analysis method (Maxam & Gilbert method) or a chain terminator method (Sanger dideoxy method).

[0064] The mutation in the gene that causes Hsp70 deficiency can be clarified by comparing base sequences of Hsp70 genes of normal bovine and attacked bovine. That is, the mutation that causes the disease can be identified by examining the base sequence of attacked bovine Hsp70 gene as in the normal bovine and comparing it with the base sequence of the normal bovine gene.

[0065] The mutation that a portion of 1997-11030 position in the base sequence of the translated region encoding Hsp70 in normal bovine Hsp70 gene shown in SEQ ID No. 1 of SEQUENCE LISTING is deficient in Hsp70 gene of Hsp70 deficiency-attacked bovine has been found in the invention (FIG. 1(a)).

[0066] In FIG. 1(a), both HSPA1A and HSPA1B are Hsp70 genes. The Hsp70 genes are two genes of almost the same sequences which are laterally arranged on a chromosome. Further, the 11-kb deficient site starts from the 3′-untranslated region of HSPA1A and ends with the 3′-terminal translated region of HSPA1B.

[0067] According to the invention, bovine Hsp70 deficiency and its carrier can be detected and diagnosed by a genetic engineering method easily and quickly. Further, a kit used for this purpose is provided.

EXAMPLES

[0068] The invention is illustrated more specifically below by referring to Examples. However, the invention is not limited thereto. In the following description, the operation is performed by a method described in D. M. Glover & B. D. Hames (Ed.), DNA Cloning 1, Core Techniques (2nd edition), A Practical Approach, Oxford University Press, Oxford (1995); J. Sambrook, E. F. Fritsch & T. Maniatis (Ed.), Molecular Cloning, A Laboratory Manual (2nd edition), Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989); M. A. Innis, D. M. Gelfaud, J. J. Snindky & T. J. White (Ed.), PCR Protocols: A Guide To Methods and Applications, Academic Press, Inc., New York (1990); M. J. McPherson, P. Quirke & G. R. Taylor (Ed.), PCR; A Practical Approach, IRL Press, Oxford (1991), or its modified or altered method, unless otherwise instructed. When a commercial kit or measuring device is used, the operation is performed according to instructions of protocols attached thereto, unless otherwise instructed.

Example 1

[0069] (1) Determination of a Base Sequence of Normal Bovine Hsp70 Gene

[0070] With respect to a family consisting of Hsp70 deficiency-attacked bovine (12 heads) and parents/daughter bovine thereof, a region of bovine chromosome linked with Hsp70 deficiency was determined using a polymorphic DNA marker. That is, a DNA marker linked most strongly with the disease among DNA markers present on a linkage map was selected. A bovine BAC (bacterial artificial chromosome) clone present in this region was separated from BAC library [manufactured by CHILDREN'S HOSPITAL OAKLAND—BACPAC RESOURCES].

[0071] Shotgun base sequence determination was performed using this BAC clone as a material. First, DNA of the BAC clone was physically cut to a size of approximately 700 bp with a nebulizer, and both ends were blunted with DNA polymerase. The cut fragments were cloned in a plasmid. Arbitrary selection was performed from these plasmid clones, and the base sequence of each of the selected clones was determined from both ends with BigDye Terminator Cycle Sequencing Reagent (manufactured by PE Biosystems) using a 3700 fluorescent DNA sequencer (manufactured by PE Biosystems). The resulting base sequence is shown in SEQ. ID No. 1 of SEQENCE LISTING. This base sequence was compared with another known gene (gene with the base sequence determined as registered in database of GenBank). Consequently, it was found to be Hsp70 gene.

[0072] (2) Expression of Hsp70

[0073] A soluble fraction as a supernatant obtained by centrifugation of a mashed muscle was prepared from a diaphragmatic muscle of deficiency-attacked bovine, and the expression of Hsp70 was examined in a usual manner. That is, western blotting was performed such that the soluble fraction was subjected to electrophoresis on a 10%-polyacrylamide gel containing SDS and blotted on a nitrocellulose membrane and expression of Hsp70 was examined using an anti-Hsp70 antibody (manufactured by Santa Cruz Biotechnology). Consequently, it was found that Hsp70 was little expressed in deficiency-attacked bovine in comparison to normal bovine.

[0074] (3) Determination of a Base Sequence of Hsp70 Gene of Hsp70 Deficiency-Attacked Bovine

[0075] As described in (2) of Example 1, Hsp70 was little expressed in Hsp70 deficiency-attacked bovine, and the base sequence of Hsp70 gene in deficiency-attacked bovine was determined from this fact. That is, PCR was performed by referring to the sequence of SEQ. ID No. 1 of SEQUENCE LISTING to determine the DNA base sequence of Hsp70 gene in Hsp70 deficiency-attacked bovine.

[0076] The thus-determined base sequence was compared with the foregoing base sequence determined in normal bovine. Consequently, in Hsp70 gene of Hsp70 gene deficiency-attacked bovine, approximately 11 kb including 1997-11030 position was deficient in the base sequence shown in SEQ ID No. 1 of SEQUENCE LISTING. For this reason, it is considered that the expression of Hsp70 is not observed in deficiency-attacked bovine.

Example 2

[0077] Detection of Hsp70 Normal Type:

[0078] In order to identify a normal type of Hsp70 in bovine genomic DNA, primers F1, R1, F2 and R2 (SEQ ID Nos. 2 to 5 of SEQUENCE LISTING) for amplifying a DNA fragment including a deficient site found in Hsp70 deficiency-attacked bovine were synthesized according to the base sequence of SEQ. ID No. 1 of SEQUENCE LISTING. The positions of these primers in the base sequence are shown in FIG. 1(b).

[0079] Genomic DNAs were prepared from blood of normal bovine, muscle of Hsp70 deficiency-attacked bovine and blood of mother or daughter bovine thereof (containing EDTA and heparin as an anticoagulant) using QIAamp Blood Kit or QIAamp Tissue Kit (manufactured by QIAGEN).

[0080] PCR was conducted using these genomic DNAs as templates, primers F1 and R1, and primers F2 and R2, and Animal Taq. In PCR, a cycle including steps at 94° C. for 20 seconds, at 60° C. for 30 seconds and at 72° C. for 1 minute was repeated 35 times. The reaction solution was subjected to electrophoresis using a 1.5% gel (1×TBE), and the gel after the electrophoresis was stained with ethidium bromide to identify the amplified DNA fragments.

[0081] When the genomic DNAs of normal bovine and mother bovine or daughter bovine of Hsp70 deficiency-attacked bovine were used as templates, the amplifications of 422-bp and 198-bp DNA fragments were observed as shown in FIG. 2(a). However, when the genomic DNA of Hsp70 deficiency-attacked bovine was used as a template, no amplification was observed.

Example 3

[0082] Detection of Hsp70 Mutant Type:

[0083] In order to identify the presence of Hsp70 gene mutant type in bovine genomic DNA, primers F3, F4 and R3 (SEQ ID Nos. 6 to 8 of SEQUENCE LISTING) were synthesized (FIG. 1(b)) on both ends of an approximately 11-kb deficient portion described in (3) of Example 1, and subjected to experiment.

[0084] PCR was conducted using genomic DNAs of normal bovine, Hsp70 deficiency-attacked bovine and mother bovine or daughter bovine thereof as templates, primers F3 and R3, and primers F4 and R3, and Animal Taq. In PCR, a cycle including steps at 94° C. for 20 seconds, at 60° C. for 30 seconds and at 72° C. for 1 minute was repeated 35 times. The reaction solution was subjected to electrophoresis using a 1.5% gel (1×TBE), and the gel after the electrophoresis was stained with ethidium bromide to identify the amplified DNA fragments.

[0085] When the genomic DNAs of Hsp70 deficiency-attacked bovine and mother bovine or daughter bovine thereof were used as templates, the amplifications of 2028-bp and 1989-bp DNA fragments were observed as shown in FIG. 2(b). However, when the genomic DNA of normal bovine was used as a template, no amplification was observed.

[0086] These results reveal that with respect to the deficiency of approximately 11 kb including the translated region of Hsp70 gene, the mother or daughter bovine of the deficiency-attacked bovine is a heterozygote to this mutation and the Hsp70 deficiency is a hereditary disease with autosome recessive heredity.

Sequence Listing

[0087] <110> Japan Livestock Technology Association

[0088] <120> Gene Diagnosis for Bovine Hsp70 Deficiency

[0089] <130> P141292K

[0090] <160>8

[0091] <210>1

[0092] <211>12988

[0093] <212>DNA

[0094] <213>Bovine

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[0098] cgtaggggtg ttccagcacg gcaaggtgga gatcatcgcc aacgaccagg gcaaccgcac 180

[0099] cacccccagc tacgtggcct tcaccgatac cgagcggctc atcggcgatg cggccaagaa 240

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840 gcgggccgtg aggcggctgc gcaccgcatg cgagcgggcc aagagaacct tgtcgtccag 900 cacccaggcc agcctggaga tcgactccct gttcgagggc atcgacttct acacgtccat 960 caccagggcg cggttcgagg agctgtgctc cgacctgttc cggagcaccc tggagcccgt 1020 ggagaaggcg ctacgcgacg ccaagctgga caaggcgcag atccacgacc tggtcctggt 1080 ggggggctcc acccgcatcc ccaaggtgca gaagctgctg caggacttct tcaacgggcg 1140 cgacctcaac aagagcatca accccgacga ggcggtggcg tacggggcgg cggtgcaggc 1200 ggccatcctg atgggggaca agtcggagaa cgtgcaggac ctgctgttgc tggacgtggc 1260 tcccctgtcg ctgggactgg agacggccgg aggcgtgatg accgccctga tcaagcgcaa 1320 ctccaccatc cccacgaagc agacgcagat cttcaccacc tactcggaca accagccggg 1380 cgtgctgatc caggtgtacg agggcgagag ggccatgacg cgggacaaca acctgctggg 1440 gcgcttcgag ctgagcggca tcccgccggc cccgcggggg gtgccccaga tcgaggtgac 1500 cttcgacatc gacgccaatg gcatcctgaa cgtcacggcc acggacaaga gcacgggcaa 1560 ggccaacaag atcaccatca ccaacgacaa gggccggctg agcaaggagg agatcgagcg 1620 catggtgcag gaggcggaaa agtacaaggc ggaggacgag gtccagcgcg agagggtgtc 1680 tgccaagaac gcgctggagt cgtacgcctt caacatgaag agcgccgtgg aggatgaggg 1740 gctgaagggc aagatcagcg aggcggacaa gaagaaggtg ctggacaagt gccaggaggt 1800 gatttcctgg ctggacgcca acaccttggc ggagaaggac gagtttgagc acaagaggaa 1860 ggagctggag caggtgtgta accccatcat cagcagactg taccaggggg cgggcggccc 1920 cggggctggc ggctttgggg ctcagggccc taaagggggc tctgggtctg gccccaccat 1980 tgaggaggtg gattaggaat ccttccctgg attgctcatg tttgttatgg agactgttgg 2040 gatccaaggc tttgcattgc cttatatatc ttcctttcat cagccatcag ctatgcaagc 2100 tgtttgagat gttgaactgt cccttttatg aaattaggaa ctcttttttc cagagtctta 2160 agtatagagc tgaatgtata gtgccatctt ttgtcagttt ctttttgtag tattcatgcc 2220 aaactcaagc tatttttcac ccgtttctgt ttacttccaa gtaaataaac tcaaataatt 2280 cgagtgatgt ttgcttctgt gtttttattt tgaagttaga aggatctgta gaggttgtct 2340 gttttacagt atccaaaaat gaactgcaat tggcctctta gataaggtca gggatccaga 2400 aaagaataca gcattatgac acatttcttt taggcaaata gtatccttgg gaaacataaa 2460 gctgctcatt tgaatggttg tgtttgtgaa tccagaaaat gttaagggtt actggcatgg 2520 tagcctcaag gttgggcggg gggtccatac tttacgggtg aactcaaaag gtgcctgtag 2580 tggcagtatt cctggagaag caggcaaata agaggcagtt agattggaag tcatgggtgc 2640 tgctgcttgt tagtacaggt gataccttag agccttgtta cttaatctag attcagcatg 2700 aaagagaagg tgagtcctaa attggcactg aggaaatgtg aattctagta ctggcttgcc 2760 taattatgca tgattgcgtt agccactgtg atcctcaagt ctcacagttt aaaatggaag 2820 ggtttggcct gatgctaaag tttaatttct taaaagaatg ctgagataaa aatgctgcgt 2880 ttccagtact ggttacctac attttaagta tcccagtgag taccttagag aggtgtcact 2940 gtttcatgcc ccagcaggag gacggacccc cagtatttca gtgtgcttac ctaccaggta 3000 ctgtaccagg ggccttttac atgtttatta attcccattc caccatattg agtataggca 3060 gtgtttggct tccacaggtg gacgtatgtg gagacttaaa aggcactggc ttaaatttat 3120 tacaagggta aaaaaacggg ttcagggaag atgttgaacc tggattccaa ctgaggtttt 3180 attgtttttt gctctgctgc ccacagggct ttgtgcatgt ctggttctgg gtctacccta 3240 ggtttcacaa tcggtaatct ttctgctttg acaatgtata atcctaaaca actatgtcag 3300 ataatacggt taatgctaga ggtttaatac tggttaattt agaagagtga ttgaaaaaac 3360 ctgcagcact gcaccaggaa gccttaacca caggcttcct tcccctgcag atgcttcttg 3420 ctttaactgt tgctagaatt ctgggaagag tcccctccac agcctgtttg tgggaaaagg 3480 cctggcacaa tcctcacgac ttggggagtg agccccttta aaaggcaatt ttatctgggg 3540 attacagaga ttctggaacc aggtggaagt ggtgattgca caaactgggc tagggaccac 3600 taaattctac actttaaaat ggtttatgtg aattcaccaa aagtagtttt taaaaaaaaa 3660 ttgtgtcaac attctggaaa aacactttgt gagtgtgtgt atctcaaggc ccaccaaatc 3720 tttcactaaa tacttgcatt agaagaaact cttaatggta ataacatgta gaggtagacc 3780 tgtccctgta agtttggaaa tggaaatcta agagatgctt agacttgcag gccagcatat 3840 aaacacaggt ttaatcctca gggtaggtga actgtagcac ggtggactgt agccacaatg 3900 tgagtcaccc ttcatgggga tatgcggttg gaacacgacc tcctctaccc ccacagaact 3960 gcagtaccat ctgtgactgt catctgcaga taatacaata actcttgaag cagtcaccct 4020 actttagggg gaggtggcaa gggatgggga gggtggggtg gagattggga aagacctaac 4080 aaacaccttt gataagagag attagggaaa tctccagaaa ttaatttgga gaaaatgagt 4140 tcctatggct aaaccagtta agattatcag ggtgttttat taggaagtca atatataatg 4200 ttactgcaca gtcccttgca cagactactt tgaaaataat caccttcaac atgaagctga 4260 gggacaaaga gaatgcaaag tcattcctgg agaaggtgat tgcggtagca gcaagaactc 4320 ggggtggggg tgggggggag gaggtgcatc aaggaaaaat aatggtcgat caaaaagcat 4380 ttttaaaatc taacaccttc cctaattcca atctcaccta cttccctatg ccagccctga 4440 aaaattagat tgttatggta atgtgactga ttttaaatcc aagatactac gttattaaca 4500 catagttact cctggtgttt aactggattc tgtcattaaa aatgaaaagg ataccaaagc 4560 aataacataa ttgtgagaga agtgcacaga agcatgggct ttcagttaaa ataaatggtt 4620 ttcaggtgaa aagtcaacac tggcgatttc tgagggggcg agcctcaagg taggaataag 4680 aaagggcaac tgtcatcatt ctttattcca actgatcacc ttaaatccat ccccaagggt 4740 cacccgcaaa gtatccagtg cagttcagta ggatatagca accccatcag tcctctccta 4800 actccagctc acgtagagac gttaaggggt caggtatcgc agcgaattcg ggatgccgag 4860 ccaacctgcc ccaccccacg ggcgccagta ccgcccagca ggaaatcgga ggaaagggca 4920 cggcggggaa ggagggaggg cacacaggaa atacagggta agggggcggg ggagtccaga 4980 agatcagaat caccccagag gatcttccac ctttttaccc gtccagacgt ccccaggaga 5040 gccagggact agattcggga gatgggacgg cggcagagag aagacagcaa gctcccagct 5100 gtagccaatc cctgcccagg gctgcggctc acccgcctct ggcggtgggg accttctagc 5160 ttctggcaac cccaatccat ccgacttact tgtgtcagtt acaaacctgt ccagtgtttt 5220 cacccaacat attagcgagt ttgagggaaa ctctaaaggt ctctccttta ctgactcctt 5280 taatcccatt ttgaaaaaga accgaagaac gccggcaccg gccaggcaac tccgcggcca 5340 gccccgccgt caggccccgc cccgctccat cggggtctta ctcgctctgg ctccttgccc 5400 ccgtttcggg ctgtgtcagg aactttctgg agctctctgg gctcagaggc ggggactggc 5460 tcgtaggaac actcttcaac aaacaaactg ccccacccaa gtctccctcc cttcctctgt 5520 taacagccga ccagtctgtg ataacgggaa ggggagacgg tcctgggaga acctggaagg 5580 gccgaaaagg tggaagtgtg ggtgttgtcg ggggaagcgg cggagctggg ggtgcgtaga 5640 taggcgtgag tcagaagcaa cagcctggag gtgagtctcc gcaggtcaca cacccccatg 5700 gtgcacgtag agccctggca ttcactcttt actgtcgtcc atggttgttt ctgttcttct 5760 tttatagagc gtggaacgat agggtttatg tgccagcatt gagaggagtc caaagtagaa 5820 agtatgccga catgttagtt caatcaccgg ttccgtaatt acctgtctgg gtgatctggc 5880 caagccacga aacctctgaa cctttgtgct catctttgaa aacagaaagg tttggctgaa 5940 ggactctgcc taaaaatctg aagatagttt ttatggtaaa ccgaaagtat tactatcata 6000 gtcctggtag taatccccaa ccttgtaagc acctcagtaa gaaatgattg agagatgaga 6060 ctcgagagag tgttacttca ataaaagaat gaagggcaca aacttttgag tacaactctg 6120 tcacagccac tgaactagtc ttttaaatat tgtctttgta atccttgatg gtatcatact 6180 atgaaataaa tattaattct aatttataca acttgtgtaa tttagttcat ttacacgtac 6240 ttcattgtta agaaagaaaa acagcttcaa caaggagata gagtccagat acaaacccag 6300 gtcttgcctt tcccagtttt ttcccccatg ctgctggaaa ttagcagagt tcccaggcct 6360 ttgccacact tccctggtgg atcagagggt gaagaatctg cccacagtgc aagagacctg 6420 ggttctatcc ctgagtagag aagatcccct ggagaaggga atggcgaccc actccagtgt 6480 tcttgtgtgg aaaatcccat gggcagagga gcctggccgg ctacagtcca cggggtcaca 6540 aaggagtcgg acatgactgg gtgactaaca ctgtcaggcc tttgcccttt gaaggttaca 6600 aatgcctggc tcagggctcg cctggtggct catcggtaaa gaatccgcct gccaatgcag 6660 gagacacagg ttctattcct gatccaggaa gattcccaca tgtcctcgtt ccaaggagca 6720 gctaagcctg tgtgccacaa ctattgagca cgtacagccc attctctgaa acaagagaag 6780 ccaccacaat gagaagcctg cttaccccca actcaactag agaatagcct ctgctcacca 6840 caactagaga aaagcctctg tagcagcaga gatctagcac agccaaaaat aaaatgaaaa 6900 aatgcctggc tctaggtgtc acattgttct cttttgcttc tgtctgaaaa acctagaatt 6960 atactgtctt ttaaaaacaa atagacttga gaaaaaccat actagatgaa aaactgtagg 7020 aaaaaggaga gagaacaaaa aaagatcctg caacttcagg gtgaggacgg ctccccccgc 7080 cccacccact tccttccctt ggcagttagc attcttggca gtctctctcc catccccaac 7140 ccttaaattt taccctgtca cccggtcagg cttgggcaac cttaatcttg attcttccaa 7200 acactaaacc cgattttaaa aaactaattc caaaatgcat caaataaagt tgtgaaaagt 7260 ctcttgggat tcttaaaatc tccttgctgc tgctgctact aagtcgcttc agttgtgtcc 7320 aactctgtgc aaccccacag acggaagccc accaggctcc ccaatccctg ggattctcca 7380 ggcaagaaca ctggagtggg ttgccatttc cttctccaat gcatgaaagt gaaaagtgaa 7440 agtgaagttg ctcaggagtc cgactcttag cgaccccatg gactgcagcc taccaggctc 7500 ctccgttcat gggattttcc aggcaagaac actggagtgg gttgccattg ccttctagag 7560 ttacactatt acactcattg atcatatatc gaactataca tttgatcaac tgcttcaagt 7620 ctagtcatca tttctgttga aagctcagtc atatacttgg taatacaaga aataataatc 7680 ttgtgaaaca agcaaaatac aaatggtata gttaataaca ttagtggaac taaaaggaga 7740 tattttagcc atgagcctcc cacaccagtt ttttttaaag attgtcaaga ctagggaatg 7800 ggtacttaga gcagaaatct gatttttcat gtggttcaaa tgtgttacat taaaggattt 7860 atcaggtaca aaaatacagc attcagtttg aattatagca cagctatctc cctgagatgc 7920 tgtcaagagt cttgcagttg tgtagcaggg cctttctcat tatagagatc tcagaagtca 7980 ataggtgaat agcctgatta tcatttaaag cttatgaaag ttgttaaggc ttagatatgg 8040 tcaattacat cctccaaccc cattgaaggc atgcacacgc gtgcgcacgc gcgcacacac 8100 acacacacac acacacacgc tgctaaatgg tcatacacca aatctcctta ggcaccaatt 8160 aaaccggtac ctgagttcct gccttgggaa gtgtccagtg ttaaaggaag acaaaattca 8220 agagactctc ctcataggaa atggaaaaga aatacggata tttaggtttc cgggtcatcc 8280 acagagagag acaacgcaaa gtgtaggtta atacagtgtg tagctgactg cttgattcat 8340 gaaaaacagc attttcaagt ggctccccca ctcctccacc ccagcaacag caagatttga 8400 ggccctatca cctgtctccc tgtcgagcag tggagacaat gatgcccttt gcttcaagcc 8460 aatagaggaa gagaactgca aattttggag aggagagcga atccagaatt cctgctggta 8520 gcagctgatg ggggagaagg caatggcaac ccactccagt gttcttgcct ggagaatccc 8580 agggacgggg gagcctggtg ggctgctgtc tctggggtcg cacagagtcg gacacaactg 8640 aagtgactta gcagtagcag cagcagctga tggtgaggaa gacaggggag aggggatgag 8700 gttaaggact tctctggagg tgaacacttc tctggaagtg ttcacaaact gggtggctaa 8760 gatggacgtt tggggaatcc cctttcagat actgcataaa gagatggaaa attcctgaag 8820 tttaaccagt ttgactagat taaggaggtg attcattgga gagccacacc tgaatgtaaa 8880 aaaagttatc acctacctgc acagtgaaag ataaaaatat tgctttaaca aatctgtata 8940 tctgattaac ctgaacaaat tataaaataa actgaatacc ctcagatttc aggaagaggt 9000 gtttgatgaa tggctgtgcg cgcgcgcgcg cgtgtgtgtg tacgtgtgta aacgtcagtt 9060 aagcaaaagt gttcaaagcg agatttcttc cctttatcag aaattgcctc ctcaggtact 9120 tctctggtgg tccagaaggg ctaagactct gtagaggaga atgcaggcgg cctgggttcg 9180 atctctggtc aagaaaatag atcccacatg ctacaactaa gattgaccat gctacaacta 9240 aggcttagct attaatttta aaacaacaac aacaaaaccc cacaactgcc tcctccgact 9300 tgtgctgtta tgttttctat gctcaagaca tgtggataca gtaatgagtc tatttcatgg 9360 gttgtgaatc ccctctacta tggctttaat gtccctcaca ttttcacttt aggtgcctaa 9420 taagggatct tgcattgccc ataaaggaag aagaaacaaa agccaaaata aattaccaaa 9480 tgtcactgta tttaaaacag gaaggaggct aacaacagaa agctgaaatc taggataaaa 9540 agttaaatgg acgaattaag tacacagcaa acaacctgaa cttttagagg agatagaacc 9600 taggtcctgc caacctttct caccttccag catcattcca gactgtttac aatgggccac 9660 ccgccaacca actatatagc atgctcttca aacaggactg aacgctcccc cacccccacc 9720 ctcgcaggct caccaccaca ccacatttac ttaaaagtag tggacagcct aggagccgca 9780 aatgacaagg cagaagaccg aattcgggac tcaggttaat ccaggcacca ctgatcatcc 9840 gaggctgaac caggaattta aaaggcacag aggaggggag gggtgcgtcc gcacctgggg 9900 ctgggaaaga tgaggaatcc ggagaagcgc aaaggacagc taaatatcta tggaaaatat 9960 tttctttctc aagcccagtc cagcccgagg agaaagggag cagctctggg cggggacagg 10020 ggcgctgtgg ctccagccct gcccttccca cgctcccccg accgagcagg tcccttctaa 10080 ggcgttggga accttctaca atctaaaaac catataccta attgattttc ttctgaaaat 10140 taaaatttcc cctcccatct gaatagggct aaagaggagc caaaacttaa acagcttcaa 10200 ctctctcctt ttccttccca ttttaaaaat aagatgggaa aagcgccgcg gatgaccaag 10260 gcatttctcg gacagcccgg ccgctcggcg agccagccca aacgtggctg cttccatcag 10320 cgttagcctc cgatcactct ccttggccca cagatagcca accctcttcg agaaactcgg 10380 gaactttctg tattttggct gtcccggcag tcgtgtagcc cttaattcta ctttaaacca 10440 ccaaactaat ttgagccccg agatcctctc accgccctac aattaattac aagcccaggg 10500 ctgatccttc cagtcgactc caaactactt ggctggctgg tcgccaggaa accagagaca 10560 gagtgggtgg accttcccag cccctctccc cctctcctta ggactcctgt ttcctccagc 10620 gaatcctaga agagtctgga gagttctggg aggagaggca tccagggcgc tgattggttc 10680 cagaaagcca gggggcagga cttgaggcga aacccctgga atattcccga cctggcagcc 10740 ccactgagct cggtcattgg ctgacgaagg gaaaaggcgg cggggcttga tgaagaatta 10800 taaacacaga gccgcctgag gagaaacagc agcctggaga gagctgataa aacttacggc 10860 ttagtccgtg agagcagctt ccgcagaccc gctatctcca aggaccgccc cgaggggcac 10920 cagagcgttc agttttcggg ttccgaaaag cccgagcttc tcgtcgcaga tcctcttcac 10980 cgatttcagg tttgaagctt atctcggagc cggaaaagca gggcaccggc atggcgaaaa 11040 acacagctat cggcatcgac ctgggcacca cctactcctg cgtaggggtg ttccagcacg 11100 gcaaggtgga gatcatcgcc aacgaccagg gcaaccgcac cacccccagc tacgtggcct 11160 tcaccgatac cgagcggctc atcggagatg cggccaagaa ccaggtggcg ctgaacccgc 11220 agaacacggt gttcgacgcg aagcggctga tcggccgcaa gttcggagac ccggtggtgc 11280 agtcggacat gaagcactgg cctttccgcg tcatcaacga cggagacaag cctaaggtgc 11340 aggtgagcta caagggggag accaaggcgt tctacccgga ggagatctcg tcgatggtgc 11400 tgaccaagat gaaggagatc gccgaggcgt acctgggcca cccggtgacc aacgcggtga 11460 tcaccgtgcc ggcctacttc aacgactcgc agcggcaggc caccaaggac gcgggggtga 11520 tcgcggggct gaacgtgctg aggatcatca acgagcccac ggccgccgcc atcgcctacg 11580 gcctggacag gacgggcaag ggggagcgca acgtgctcat ctttgatctg ggagggggca 11640 cgttcgacgt gtccatcctg acgatcgacg acggcatctt cgaggtgaag gccacggccg 11700 gggacacgca cctgggcggg gaggacttcg acaacaggct ggtgaaccac ttcgtggagg 11760 agttcaagag gaagcacaag aaggacatca gccagaacaa gcgggccgtg aggcggctgc 11820 gcaccgcatg cgagcgggcc aagagaacct tgtcgtccag cacccaggcc agcctggaga 11880 tcgactccct gttcgagggc atcgacttct acacgtccat caccagggcg cggttcgagg 11940 agctgtgctc cgacctgttc cggagcaccc tggagcccgt ggagaaggcg ctacgcgacg 12000 ccaagctgga caaggcgcag atccacgacc tggtcctggt ggggggctcc acccgcatcc 12060 ccaaggtgca gaagctgctg caggacttct tcaacgggcg cgacctcaac aagagcatca 12120 accccgacga ggcggtggcg tacggggcgg cggtgcaggc ggccatcctg atgggggaca 12180 agtcggagaa cgtgcaggac ctgctgttgc tggacgtggc tcccctgtcg ctgggactgg 12240 agacggccgg aggcgtgatg accgccctga tcaagcgcaa ctccaccatc cccacgaagc 12300 agacgcagat cttcaccacc tactcggaca accagccggg cgtgctgatc caggtgtacg 12360 agggcgagag ggccatgacg cgggacaaca acctgctggg gcgcttcgag ctgagcggca 12420 tcccgccggc cccgcggggg gtgccccaga tcgaggtgac cttcgacatc gacgccaatg 12480 gcatcctgaa cgtcacggcc acggacaaga gcacgggcaa ggccaacaag atcaccatca 12540 ccaacgacaa gggccggctg agcaaggagg agatcgagcg catggtgcag gaggcggaaa 12600 agtacaaggc ggaggacgag gtccagcgcg agagggtgtc tgccaagaac gcgctggagt 12660 cgtacgcctt caacatgaag agcgccgtgg aggatgaggg gctgaagggc aagatcagcg 12720 aggcggacaa gaagaaggtg ctggacaagt gccaggaggt gatttcctgg ctggacgcca 12780 acaccttggc ggagaaggac gagtttgagc acaagaggaa ggagctggag caggtgtgta 12840 accccatcat cagcagactg taccaggggg cgggcggccc cggggctggc ggctttgggg 12900 ctcagggccc taaagggggc tctgggtctg gccccaccat tgaggaggtg gactaggggc 12960 cttacttttt gtctgtctgt agtagacc 12988 2 20 DNA Artificial Sequence Oligonucleotide to act as a primer for PCR 2 aaccccatca tcagcagact 20 3 21 DNA Artificial Sequence Oligonucleotide to act as a primer for PCR 3 cacagaagca aacatcactc g 21 4 20 DNA Artificial Sequence Oligonucleotide to act as a primer for PCR 4 gcattgccca taaaggaaga 20 5 20 DNA Artificial Sequence Oligonucleotide to act as a primer for PCR 5 tggaaggtga gaaaggttgg 20 6 19 DNA Artificial Sequence Oligonucleotide to act as a primer for PCR 6 acgtcgttga tcctgtggg 19 7 19 DNA Artificial Sequence Oligonucleotide to act as a primer for PCR 7 tatctcggag ccgaaaagg 19 8 29 DNA Artificial Sequence Oligonucleotide to act as a primer for PCR 8 ggtctactac agacagacaa aaagtaagg 29 

What is claimed is:
 1. A method for gene diagnosis of bovine Hsp70 deficiency, which comprises the following steps, (a) a step of obtaining a bovine nucleic acid sample, (b) a step of subjecting the nucleic acid sample obtained in step (a) to a gene amplification reaction to obtain a nucleic acid fragment in which a region including a mutation site likely to be present in bovine Hsp70 gene is amplified, and (c) a step of examining the presence of mutation on the nucleic acid fragment in step (b), the region including the mutation site being a region including 1997-11030 position of a base sequence shown in SEQ ID No. 1 of SEQUENCE LISTING in a base sequence of bovine Hsp70 gene.
 2. The method for gene diagnosis as claimed in claim 1, wherein the gene amplification reaction is conducted by a polymerase chain reaction method.
 3. The method for gene diagnosis as claimed in claim 1 or 2, wherein the presence of mutation is examined by examining a gene amplification product obtained by the polymerase chain reaction method.
 4. The method for gene diagnosis as claimed in any one of claims 1 to 3, wherein the nucleic acid sample is a sample containing genomic DNA, cDNA or mRNA.
 5. A method for gene diagnosis of bovine Hsp70 deficiency, which comprises conducting genome linkage analysis of subject bovine, isolating bovine Hsp70 gene by positional cloning, determining a base sequence of the gene by a usual manner, and examining the presence or absence of mutation by comparing said base sequence with a base sequence of cDNA encoding normal bovine Hsp70 as shown in SEQ ID No. 1 of SEQUENCE LISTING.
 6. A kit for detecting bovine Hsp70 deficiency, which kit contains oligonucleotide primers used for amplifying a region including a mutation site likely to be present in bovine Hsp70 gene by a gene amplification reaction, the oligonucleotide primers being selected from the group consisting of (1) oligonucleotides having a base sequence corresponding to a 5′-terminal region in a base sequence shown in SEQ ID No. 1 of SEQUENCE LISTING, and (2) oligonucleotides having a complementary base sequence to a 3′-terminal region in the base sequence shown in SEQ ID No. 1 of SEQUENCE LISTING.
 7. The kit as claimed in claim 6, wherein the oligonucleotide primers comprise from 15 to 35 nucleotides.
 8. The kit as claimed in claim 6, wherein the oligonucleotide primers are a pair of oligonucleotide primers selected from the group consisting of those shown in SEQ ID Nos. 2 to 8 of SEQUENCE LISTING, provided combinations of SEQ ID Nos. 2 and 4, 3 and 5, and 6 and 7 are excluded. 